More than 10% of Americans suffer from chronic sleep disorders, with an estimated annual cost of $100 billion and for which few therapies are available. Despite the impact of sleep disorders, the fact that we spend a third of our lives asleep, and the evolutionary conservation of sleep-like states, mechanisms that regulate sleep are poorly understood. Progress in understanding these mechanisms in vertebrates has been hindered in part by the complexity of mammalian brains and the challenge of performing genetic screens in mammals. To overcome these limitations, we and others recently demonstrated behavioral, anatomical, genetic and pharmacological conservation of sleep between zebrafish and mammals, establishing zebrafish as a simple and inexpensive vertebrate model for sleep. We recently used zebrafish to perform the first large-scale screen for genes that regulate vertebrate sleep. We found that overexpression of the neuropeptide neuromedin U (Nmu) promotes locomotor activity and inhibits sleep. Most studies of Nmu in mammals have focused on its role in regulating appetite, although acute Nmu administration has been shown to transiently prolong wakefulness and disrupt sleep. However, a role for Nmu in regulating sleep has not been extensively studied and the potential role of nmu-expressing neurons in sleep has not been explored. The objective of this proposal is to use zebrafish to determine the genetic and neurological mechanisms through which Nmu and nmu-expressing neurons regulate sleep. In Specific Aim 1 we will test the hypothesis the nmu and its receptors are required for normal levels of wakefulness by generating mutations in each gene using CRISPR/Cas9- mediated genome editing and monitoring effects on sleep using high-throughput behavioral assays. We will also investigate genetic mechanisms that mediate the effects of Nmu on sleep by mating Nmu gain-of-function and loss-of-function zebrafish to a collection of mutant and transgenic lines that we have generated that lack or allow overexpression of neuromodulators and neuropeptides that affect sleep in zebrafish and mammals. These experiments will place Nmu signaling in the context of known sleep regulatory mechanisms. In Specific Aim 2 we will test the hypothesis that nmu-expressing neurons promote wakefulness and inhibit sleep by stimulating, inhibiting and ablating these neurons using high-throughput optogenetic and chemical-genetic methods that we recently developed. In Specific Aim 3 we will test the hypothesis that Nmu promotes wakefulness by stimulating corticotropin releasing hormone (crh)-expressing brainstem neurons, and that Crh signaling, the locus coeruleus and noradrenaline are required for Nmu-induced arousal. Validation of this hypothesis would identify a novel arousal promoting neuronal circuit. By characterizing a gene and neuronal circuit that regulate sleep, our results may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neurological disorders, and may be causal in some cases, this project may also eventually lead to improved therapies for some neurological disorders.